Protocol

Introduction of Caged Peptide/Protein into Cells Using Bead Loading

This protocol was adapted from "Application of Light-Directed Activation of Caged Biomolecules and CALI to Problems in Cell Motility," Chapter 10, in Live Cell Imaging (eds. Goldman and Spector). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.

INTRODUCTION

Activation and inactivation of proteins using photoactivation of caged peptides or proteins offers insights into cellular dynamics not achievable using genetic means. The ability to selectively alter the activity of a specific protein at a defined time and location inside a cell allows the correlation of changes in protein activity and cellular behavior. A caged compound, peptide, or protein is prepared by covalently linking it to a photolabile, protecting group via a limited number of critical functional groups in the biomolecules. Under carefully defined conditions, a brief irradiation with UV light (320-400 nm) can lead to an intramolecular photoisomerization reaction that induces the cleavage of the chemical bond between the cage group and the unmodified, biologically active species. Three strategies can be used to introduce caged compounds into cells: cell-permeant peptide vectors, bead loading, and microinjection. In bead loading, cells are bathed in a medium containing the peptide/protein of interest. Glass beads are then sprinkled onto the cells. The beads create temporary holes in the cell membranes, allowing some of the peptide molecules to enter cells at random. Due to the low efficiency of this technique, a large amount of protein or peptide is needed to label a relatively small number of cells. If the peptides or proteins are not fluorescent, it is useful to co-load the cells with an inert fluorescent marker, such as rhodamine dextran, to indicate successful loading. A representative bead-loading protocol using fish keratocytes is given here.